PROJECT SUMMARY Proper mitochondrial function requires the coordinated expression of 37 genes encoded in the circular genome inside the mitochondrion, and over 1000 genes encoded on nuclear chromosomes. This intracellular, intergenomic communication presents a complicated network of interacting genes that are critical for the energy production that sustains life. Because each of these genes is variable in natural populations, each gene-by-gene interaction can be altered by the variation among individuals. Moreover, the mitochondrion is a hub of many signaling pathways that sense nutrients, oxygen, redox state of the cell, and temperature making it sensitive to environmental conditions. As a result, these interactions present a complex system that lies between genotype and phenotype. The first Aim is to map specific nuclear genes that interact with mtDNA-encoded genes that jointly cause developmental delay in Drosophila. The goals of the previous cycle of funding were to identify these `mitonuclear' interactions by replacing mtDNAs from D. melanogaster or D. simulans in to multiple strains of the Drosophila Genetics Reference Panel (DGRP). This goal has been achieved, so Aim 1 tests the hypotheses that DGRP strains causing developmental delays for all `foreign' Dsim-mtDNAs harbor multiple `mitonuclear genes' affecting development, while DGRP strains causing delays for single-mtDNAs harbor single-factor mitonuclear genes. The second Aim will identify the mutations in these genes and test their direct function using transgenic rescue experiments. A second goal of the previous funding period was to determine the genotype-by-environment (GxE) interactions for mitonuclear genotypes by exposing them to altered diets. We have determined that strong mitonuclear interactions in the DGRP can be eliminated by altering the protein content of the diet. Thus, The third Aim tests the hypothesis that the mitonuclear epistatic partners affecting development time are the loci responsible for dietary modification of this trait, vs. other trans-acting factors. Gene-by-gene (GxG) and GxE interactions are fundamental components of complex phenotypes, but the mechanistic bases of these interactions are poorly understood. Because most genome wide association studies (GWAS) do not test for mtDNA or joint mitonuclear interaction effects on phenotype, our proposed experiments address this shortcoming directly, and may identify factors underlying `missing heritability' not identified in GWAS. The research is relevant to the genetic interactions important in mitochondrial replacement therapies and the biochemical pathways affecting obesity and metabolic syndromes. By manipulating dietary environments, we may identify novel roles for mitonuclear interactions relevant to pharmaceutical treatments of these conditions.